Myotonic dystrophy (DM1) is the most common adult onset muscular dystrophy in humans. Currently, there is no cure or an FDA approved drug for DM1 and related diseases. The genetic basis for DM1 is the expansion of a CTG-repeat sequence in the 3' untranslated region of the protein kinase gene, DMPK. This defect results in the expression of toxic RNAs encoding expanded CUG repeats (CUGexp) that form aberrant intranuclear RNA-protein complexes. We have developed robust high throughput screens to identify small-molecules that selectively alter the biology of CUGexp without affecting the wild-type RNA. A screen of a highly diverse, drug- like small molecule library resulted in the identification of a potent compound MDI16, which reverses DM1 pathology in patient myoblasts and in the HSALR mouse model for DM1. As small molecules that selectively reverse CUGexp RNA toxicity in vivo are rare, this molecule provides a powerful opportunity to bring to light key small molecule-CUGexp RNA interactions that facilitate rescue of DM1 pathology. In this application we propose to determine the mechanism of MDI16 action. Aim1: Identify the chemistry that forms the basis of MDI16 potency: Preliminary studies demonstrate that MDI16 efficacy and toxicity are chemically separable. Therefore we propose to use a set of MDI16 analogs to define the chemistry rules that underlie potency and toxicity over a range of pathological CTG repeat lengths. Aim2: Determine the mechanism of action of MDI16 and its most potent analogs: Using a set of mutually supporting biochemical and biophysical assays the timing, mechanism and kinetics of small molecule interaction with CUGexp RNAs will be examined. These data will be used to develop and test molecular models. Aim3: Test the efficacy of MDI16 reversal of DM1 skeletal muscle and CNS pathology and decipher the molecular basis of the rescue using transcriptome analyses: A combination of in vivo efficacy studies and transcriptome analysis will be used to define the transcriptome changes that underlie efficacy and potential toxicity in vivo. Dual molecular understanding of both chemical potency and toxicity will accelerate the development of an efficacious and safe small molecule therapy for DM1 and related disorders.